Star-PAP controls oncogene expression through primary miRNA 3'-end formation to regulate cellular proliferation and tumour formation.

Star-PAP is a non-canonical poly(A) polymerase that is down regulated in breast cancer. While Star-PAP down regulation impairs target mRNA polyadenylation, paradoxically, we see up regulation of a large number of oncogenes on Star-PAP knockdown. Using two breast cancer cells (MCF7 with high Star-PAP, and MDA-MB-231 with negligible Star-PAP level), we discover that Star-PAP negatively regulates oncogene expression and subsequently cellular proliferation. This regulation is compromised with Star-PAP mutant of 3'-end processing function (serine 6 to alanine, S6A phospho-mutation). Concomitantly, xenograft mice model using MDA-MB-231 cells reveals a reduction in the tumour formation on ectopic Star-PAP expression that is ameliorated by S6A mutation. We find that Star-PAP control of target oncogene expression is independent of Star-PAP-mediated alternative polyadenylation or target mRNA 3'-end formation. We demonstrate that Star-PAP regulates target oncogenes through cellular miRNAs (miR-421, miR-335, miR-424, miR-543, miR-205, miR-34a, and miR-26a) that are down regulated in breast cancer. Analysis of various steps in miRNA biogenesis pathway reveals that Star-PAP regulates 3'-end formation and synthesis of primary miRNA (host) transcripts that is dependent on S6 phosphorylation thus controlling mature miRNA generation. Using mimics and inhibitors of two target miRNAs (miR-421 and miR-424) after Star-PAP depletion in MCF7 or ectopic expression in MDA-MB-231 cells, we demonstrate that Star-PAP controls oncogene expression and cellular proliferation through targeting miRNAs that regulates tumour formation. Our study establishes a novel mechanism of oncogene expression independent of alternative polyadenylation through Star-PAP-mediated miRNA host transcript polyadenylation that regulates breast cancer progression.

Proto-oncogene cSrc-mediated RBM10 phosphorylation arbitrates anti-hypertrophy gene program in the heart and controls cardiac hypertrophy.

AIMS: RBM10 is a well-known RNA binding protein that regulates alternative splicing in various disease states. We have shown a splicing-independent function of RBM10 that regulates heart failure. This study aims to unravel a new biological function of RBM10 phosphorylation by proto-oncogene cSrc that enables anti-hypertrophy gene program and controls cardiac hypertrophy. MATERIALS AND METHODS: We employ in vitro and in vivo approaches to characterise RBM10 phosphorylation at three-tyrosine residues (Y81, Y500, and Y971) by cSrc and target mRNA regulation. We also use isoproterenol induced rat heart and cellular hypertrophy model to determine role of cSrc-mediated RBM10 phosphorylation. KEY FINDINGS: We show that RBM10 phosphorylation is induced in cellular and animal heart model of cardiac hypertrophy and regulates target mRNA expression and 3'-end formation. Inhibition of cSrc kinase or mutation of the three-tyrosine phosphorylation sites to phenylalanine accentuates myocyte hypertrophy, and results in advancement and an early attainment of hypertrophy in the heart. RBM10 is down regulated in the hypertrophic myocyte and that its re-expression reverses cellular and molecular changes in the myocyte. However, in the absence of phosphorylation (cSrc inhibition or phospho-deficient mutation), restoration of endogenous RBM10 level in the hypertrophic heart or ectopic re-expression in vitro failed to reverse cardiomyocyte hypertrophy. Mechanistically, loss of RBM10 phosphorylation inhibits nuclear localisation and interaction with Star-PAP compromising anti-hypertrophy gene expression. SIGNIFICANCE: Our study establishes that cSrc-mediated RBM10 phosphorylation arbitrates anti-hypertrophy gene program. We also report a new functional regulation of RBM10 by phosphorylation that is poised to control heart failure.

Essential Role of Anisotropy in Bioengineered Cardiac Tissue Models.

As regulatory bodies encourage alternatives to animal testing, there is renewed interest in engineering disease models, particularly for cardiac tissues. The aligned organization of cells in the mammalian heart controls the electrical and ionic currents and its ability to efficiently circulate blood to the body. Although the development of engineered cardiac systems is rising, insights into the topographical aspects, in particular, the necessity to design in vitro cardiac models incorporating cues for unidirectional cell growth, is lacking. This review first summarizes the widely used methods to organize cardiomyocytes (CMs) unidirectionally and the ways to quantify the resulting cellular alignment. The behavior of CMs in response to alignment is described, with emphasis on their functions and underlying mechanisms. Lastly, the limitations of state-of-the-art techniques to modulate CM alignment in vitro and opportunities for further development in the future to improve the cardiac tissue models that more faithfully mimic the pathophysiological hallmarks are outlined. This review serves as a call to action for bioengineers to delve deeper into the in vivo role of cellular organization in cardiac muscle tissue and draw inspiration to effectively mimic in vitro for engineering reliable disease models.

Sign of APOBEC editing, purifying selection, frameshift, and in-frame nonsense mutations in the microevolution of lumpy skin disease virus.

The lumpy skin disease virus (LSDV), which mostly affects ruminants and causes huge-economic loss, was endemic in Africa, caused outbreaks in the Middle East, and was recently detected in Russia, Serbia, Greece, Bulgaria, Kazakhstan, China, Taiwan, Vietnam, Thailand, and India. However, the role of evolutionary drivers such as codon selection, negative/purifying selection, APOBEC editing, and genetic variations such as frameshift and in-frame nonsense mutations in the LSDVs, which cause outbreaks in cattle in various countries, are still largely unknown. In the present study, a frameshift mutation in LSDV035, LSDV019, LSDV134, and LSDV144 genes and in-frame non-sense mutations in LSDV026, LSDV086, LSDV087, LSDV114, LSDV130, LSDV131, LSDV145, LSDV154, LSDV155, LSDV057, and LSDV081 genes were revealed among different clusters. Based on the available complete genome sequences, the prototype wild-type cluster-1.2.1 virus has been found in other than Africa only in India, the wild-type cluster-1.2.2 virus found in Africa were spread outside Africa, and the recombinant viruses spreading only in Asia and Russia. Although LSD viruses circulating in different countries form a specific cluster, the viruses detected in each specific country are distinguished by frameshift and in-frame nonsense mutations. Furthermore, the present study has brought to light that the selection pressure for codons usage bias is mostly exerted by purifying selection, and this process is possibly caused by APOBEC editing. Overall, the present study sheds light on microevolutions in LSDV, expected to help in future studies towards disturbed ORFs, epidemiological diagnostics, attenuation/vaccine reverts, and predicting the evolutionary direction of LSDVs.

G9a and Sirtuin6 epigenetically modulate host cholesterol accumulation to facilitate mycobacterial survival.

Cholesterol derived from the host milieu forms a critical factor for mycobacterial pathogenesis. However, the molecular circuitry co-opted by Mycobacterium tuberculosis (Mtb) to accumulate cholesterol in host cells remains obscure. Here, we report that the coordinated action of WNT-responsive histone modifiers G9a (H3K9 methyltransferase) and SIRT6 (H3K9 deacetylase) orchestrate cholesterol build-up in in vitro and in vivo mouse models of Mtb infection. Mechanistically, G9a, along with SREBP2, drives the expression of cholesterol biosynthesis and uptake genes; while SIRT6 along with G9a represses the genes involved in cholesterol efflux. The accumulated cholesterol in Mtb infected macrophages promotes the expression of antioxidant genes leading to reduced oxidative stress, thereby supporting Mtb survival. In corroboration, loss-of-function of G9a in vitro and pharmacological inhibition in vivo; or utilization of BMDMs derived from Sirt6-/- mice or in vivo infection in haplo-insufficient Sirt6-/+ mice; hampered host cholesterol accumulation and restricted Mtb burden. These findings shed light on the novel roles of G9a and SIRT6 during Mtb infection and highlight the previously unknown contribution of host cholesterol in potentiating anti-oxidative responses for aiding Mtb survival.

PARP1 inhibition protects mice against Japanese encephalitis virus infection.

Japanese encephalitis (JE) is a vector-borne viral disease that causes acute encephalitis in children. Although vaccines have been developed against the JE virus (JEV), no effective antiviral therapy exists. Our study shows that inhibition of poly(ADP-ribose) polymerase 1 (PARP1), an NAD+-dependent (poly-ADP) ribosyl transferase, protects against JEV infection. Interestingly, PARP1 is critical for JEV pathogenesis in Neuro-2a cells and mice. Small molecular inhibitors of PARP1, olaparib, and 3-aminobenzamide (3-AB) significantly reduce clinical signs and viral load in the serum and brains of mice and improve survival. PARP1 inhibition confers protection against JEV infection by inhibiting autophagy. Mechanistically, upon JEV infection, PARP1 PARylates AKT and negatively affects its phosphorylation. In addition, PARP1 transcriptionally upregulates PTEN, the PIP3 phosphatase, negatively regulating AKT. PARP1-mediated AKT inactivation promotes autophagy and JEV pathogenesis by increasing the FoxO activity. Thus, our findings demonstrate PARP1 as a potential mediator of JEV pathogenesis that can be effectively targeted for treating JE.

Deciphering the Origin of DNA Viruses (Replication-Associated Parvo-NS1) That Infect Vertebrates from Invertebrate-Infecting Viruses.

DNA replication is a standard and essential function among DNA viruses; however, this functional domain's common ancestor, origin, and evolutionary path in invertebrate- and vertebrate-infecting viruses are not yet fully understood. Here, we present evidence, using a combination of phylogenetic relationships, coevolution, and CLANS (cluster analysis of sequences) analysis, that the parvo-NS1 domain (nonstructural protein NS1, DNA helicase domain) of these DNA viruses that infect vertebrates potentially originated from the invertebrate (Platyhelminthes) parvo-NS1 domain of parvovirus-related sequences (PRSs). Our results suggest that papillomaviruses and the parvovirus subfamilies Densovirinae and Hamaparvovirinae DNA helicase evolved directly from the Platyhelminthes NS1 domain (PRSs). Similarly, the parvovirus subfamily Parvovirinae NS1 domain displayed evolutionary heritage from the PRSs through Hamaparvovirinae. Further, our analysis also clarified that herpesviruses and adenoviruses independently obtained the parvo-NS1 domain from Dependoparvovirus (Parvovirinae). Furthermore, virus-host coevolution analysis revealed that the parvovirus NS1 domain has coevolved with hosts, from flatworms to humans, and it appears that the papillomavirus may have obtained the DNA helicase during the early stages of parvovirus evolution and later led to the development of the DNA helicase of adomavirus and polyomavirus. Finally, herpesviruses and adenoviruses likely inherited the parvo-NS1 domain from Dependoparvovirus in the later stages of evolution. To the best of our knowledge, this is the first evolutionary evidence to suggest that the DNA helicase of viruses that infect vertebrates originated from the invertebrate PRSs. IMPORTANCE DNA replication of DNA viruses is an essential function. This allows DNA replication of viruses to form virus particles. The DNA helicase domain is responsible for this primary function. This domain is present in parvoviruses, papillomaviruses, polyomaviruses, herpesviruses, and adenoviruses. But little is known about the common ancestor, origin, and evolutionary path of DNA helicase in invertebrate- and vertebrate-infecting viruses. Here, we report the possibility of the origin of DNA viruses (DNA helicase) infecting vertebrates from Platyhelminthes (invertebrate) PRSs. Our study established that the parvovirus subfamily Parvovirinae NS1 domain displayed evolutionary heritage from the Platyhelminthes PRSs through Hamaparvovirinae. Furthermore, our study suggests that the papillomavirus DNA helicase may have evolved in the early stages of parvovirus evolution and then led to the development of the adomavirus and polyomavirus. Our study suggests that the herpesviruses and adenoviruses likely inherited the parvo-NS1 domain through gene capture from Dependoparvovirus in the later stages of parvovirus evolution in their hosts.

Unique Tandem Repeats in the Inverted Terminal Repeat Regions of Monkeypox Viruses.

The genetic diversity, especially in noncoding regions between clade I, clade IIa, and clade IIb monkeypox viruses (MPXVs), is still not fully understood. Here, we report that unique 16-nucleotide-length tandem repeats in MPXVs viruses are located in the noncoding regions of inverted terminal repeats (ITR), and the copy number of this repeat is different among clade I, clade IIa, and clade IIb viruses. It is noteworthy that tandem repeats containing these specific sequences (AACTAACTTATGACTT) are only present in MPXVs and are not found in other poxviruses. Also, the tandem repeats containing these specific sequences (AACTAACTTATGACTT) do not correspond to the tandem repeats present in the human and rodent (mice and rat) genomes. On the other hand, some of the reported tandem repeats in the human and rodent (mice and rat) genomes are present in the clade IIb-B.1 lineage of MPXV. In addition, it is noteworthy that the genes flanking these tandem repeats are lost and gained compared between clade I, clade IIa, and clade IIb MPXV. IMPORTANCE The different groups of MPXVs contain unique tandem repeats with different copy numbers in the ITR regions, and these repeats may be likely to play a role in the genetic diversity of the virus. Clade IIb (B) MPXV contains 38 and 32 repeats similar to the Tandem repeats reported in the human and rodent genome, respectively. However, none of these 38 (human) and 32 (rodent) tandem repeats matched the tandem repeats (AACTAACTTATGACTT) found in the present study. Finally, when developing attenuated or modified MPXV vaccine strains, these repeats in noncoding genomic regions can be exploited to incorporate foreign proteins (adjuvants/other virus proteins/racking fluorescent proteins such as green fluorescent protein) to carry out studies such as vaccine production and virus pathogenesis.

Sirtuin 6 inhibition protects against glucocorticoid-induced skeletal muscle atrophy by regulating IGF/PI3K/AKT signaling.

Chronic activation of stress hormones such as glucocorticoids leads to skeletal muscle wasting in mammals. However, the molecular events that mediate glucocorticoid-induced muscle wasting are not well understood. Here, we show that SIRT6, a chromatin-associated deacetylase indirectly regulates glucocorticoid-induced muscle wasting by modulating IGF/PI3K/AKT signaling. Our results show that SIRT6 levels are increased during glucocorticoid-induced reduction of myotube size and during skeletal muscle atrophy in mice. Notably, overexpression of SIRT6 spontaneously decreases the size of primary myotubes in a cell-autonomous manner. On the other hand, SIRT6 depletion increases the diameter of myotubes and protects them against glucocorticoid-induced reduction in myotube size, which is associated with enhanced protein synthesis and repression of atrogenes. In line with this, we find that muscle-specific SIRT6 deficient mice are resistant to glucocorticoid-induced muscle wasting. Mechanistically, we find that SIRT6 deficiency hyperactivates IGF/PI3K/AKT signaling through c-Jun transcription factor-mediated increase in IGF2 expression. The increased activation, in turn, leads to nuclear exclusion and transcriptional repression of the FoxO transcription factor, a key activator of muscle atrophy. Further, we find that pharmacological inhibition of SIRT6 protects against glucocorticoid-induced muscle wasting in mice by regulating IGF/PI3K/AKT signaling implicating the role of SIRT6 in glucocorticoid-induced muscle atrophy.

Evolution of monkeypox virus from 2017 to 2022: In the light of point mutations.

Monkeypox virus (MPXV) causing multi-country outbreak-2022 is related to viruses caused outbreak-2017-2018 in West Africa. Still not fully understood which proteins of the MPXV discovered in Nigeria in 2017 have mutated through different lineages to the extent that it could cause a multi-country outbreak in 2022; similarly, codon usage bias, host adaptation indices, and the role of selection or mutation pressure in the mutated genes are also not fully studied. Here we report that according to the available sequence data this monkeypox virus acquires point mutations in multiple proteins in each period, and these point mutations accumulate and become a virus that can root outbreak-2022. Viruses exported from Nigeria to Singapore, Israel, and the United Kingdom in 2018-2019 were developed as evolutionary ancestors to B.1 viruses (MPXVs causing multi-country outbreak-2022) through MPXV/United States/2021/MD virus. Although these exported viruses have different amino acid mutations in different proteins, amino acid mutations in 10 proteins are common among them. The MPXV-United Kingdom-P2 virus evolved with only mutations in these 10 proteins and further evolved into MPXV/United States/2021/MD with amino acid mutations in 26 (including amino acid mutations in 10 proteins of the MPXV-United States-P2) proteins. It is noteworthy that specific amino acid mutations in these 22/26 (presence in MPXV/United States/2021/MD) proteins are present in B.1 viruses. Further, analysis of Relative Synonymous Codon Usage (RSCU), Synonymous Codon Usage Fraction (SCUF), and Effective Number of Codons (ENc) revealed codon usage bias in genes that exhibited nucleotide mutations in lineage B.1. Also, host adaptation indices analyzes such as Codon Adaptation Index (CAI), Expected-CAI (eCAI), Relative Codon Deoptimization Index (RCDI) and Expected value for the RCDI (eRCDI) analyzes reveal that the genes that demonstrated nucleotide mutations in lineage B.1 are favorable for human adaptation. Similarly, ENc-GC3s plot, Neutrality plot, and Parity Rule 2 (PR2)-bias plot analyzes suggest a major role of selection pressure than mutation pressure in the evolution of genes displaying nucleotide mutations in lineage B.1. Overall, from 2017 to 2022, MPXV's mutation and spread suggests that this virus continues to evolve through point mutation in the genes according to the available sequence data.

Adiponectin receptor 1 variants contribute to hypertrophic cardiomyopathy that can be reversed by rapamycin.

Hypertrophic cardiomyopathy (HCM) is a heterogeneous genetic heart muscle disease characterized by hypertrophy with preserved or increased ejection fraction in the absence of secondary causes. However, recent studies have demonstrated that a substantial proportion of individuals with HCM also have comorbid diabetes mellitus (~10%). Whether genetic variants may contribute a combined phenotype of HCM and diabetes mellitus is not known. Here, using next-generation sequencing methods, we identified novel and ultrarare variants in adiponectin receptor 1 (ADIPOR1) as risk factors for HCM. Biochemical studies showed that ADIPOR1 variants dysregulate glucose and lipid metabolism and cause cardiac hypertrophy through the p38/mammalian target of rapamycin and/or extracellular signal-regulated kinase pathways. A transgenic mouse model expressing an ADIPOR1 variant displayed cardiomyopathy that recapitulated the cellular findings, and these features were rescued by rapamycin. Our results provide the first evidence that ADIPOR1 variants can cause HCM and provide new insights into ADIPOR1 regulation.

Recapitulating pathophysiology of skeletal muscle diseases in vitro using primary mouse myoblasts on a nanofibrous platform.

Tissue engineering approaches are used to mimic the microenvironment of the skeletal muscle in vitro. However, the validation of a bioengineered muscle as a model to study diseases is inadequate. Here, we present polycaprolactone nanofibers as a robust platform that mimics cellular organization and recapitulates critical functions of the myotubes observed in vivo. We isolated myoblasts from mice following a simplified protocol and cultured them on aligned nanofibers. Myotubes grown on aligned nanofibers maintained alignment for 14 days and exhibited a time-dependent increase in levels of p-AKT upon insulin stimulation. Treatment with matrix-assisted integrin inhibitor led to reduction in p-AKT levels, underscoring the critical role of environment on the biological processes. We demonstrate the suitability of myotubes grown on nanofibrous platform to study corticosteroid-induced muscle degeneration. This study, thus, demonstrates that aligned nanofibers retain myotubes in culture for longer duration and recapitulate the functions of skeletal muscle under pathophysiological conditions.

Nanostructured polymer scaffold decorated with cerium oxide nanoparticles toward engineering an antioxidant and anti-hypertrophic cardiac patch.

Reactive oxygen species (ROS) are generated in reperfused ischemic heart tissue after myocardial infarction (MI). A compensatory attempt of the heart to enhance its functional performance after MI is to undergo cardiomyocyte hypertrophy. In the past, reducing the levels of ROS in the cardiomyocytes has been linked to suppression of cardiac hypertrophy. Notably, cerium oxide nanoparticles (nCe) have been used extensively to protect the cells from oxidative damage by efficiently scavenging cellular ROS. Furthermore, fibrous matrices such as nanofibers are emerging as promising substrates for engineering implantable cardiac patches. In this study, we describe the fabrication of nCe-decorated polycaprolactone (PCL) and PCL-gelatin blend (PCLG) nanofibers prepared using electrospinning. Characterization by X-ray diffraction, X-ray photoelectron spectroscopy, energy-dispersive X-ray spectroscopy, scanning electron microscopy, atomic force microscopy, and contact angle goniometry confirmed the presence of nCe on PCL or PCLG nanofibers (PCLG-Ce) of ≈300 nm fiber diameter. nCe-based PCLG scaffolds were cytocompatible with a variety of cell types, including primary cells. Primary cardiomyocytes cultured on nCe-decorated PCLG nanofibers showed marked reduction in the ROS levels when subjected to H2O2 induced oxidative stress. Interestingly, we found that nCe-decorated PCLG nanofibers can suppress agonist-induced cardiac hypertrophy. Overall, the results of this study suggest the potential of nCe-decorated PCLG nanofibers as a cardiac patch with antioxidant and anti-hypertrophic properties.

Sirtuin 6 deficiency transcriptionally up-regulates TGF-ß signaling and induces fibrosis in mice.

Caloric restriction has been associated with increased life span and reduced aging-related disorders and reduces fibrosis in several diseases. Fibrosis is characterized by deposition of excess fibrous material in tissues and organs and is caused by aging, chronic stress, injury, or disease. Myofibroblasts are fibroblast-like cells that secrete high levels of extracellular matrix proteins, resulting in fibrosis. Histological studies have identified many-fold increases of myofibroblasts in aged organs where myofibroblasts are constantly generated from resident tissue fibroblasts and other cell types. However, it remains unclear how aging increases the generation of myofibroblasts. Here, using mouse models and biochemical assays, we show that sirtuin 6 (SIRT6) deficiency plays a major role in aging-associated transformation of fibroblasts to myofibroblasts, resulting in tissue fibrosis. Our findings suggest that SIRT6-deficient fibroblasts transform spontaneously to myofibroblasts through hyperactivation of transforming growth factor ß (TGF-ß) signaling in a cell-autonomous manner. Importantly, we noted that SIRT6 haploinsufficiency is sufficient for enhancing myofibroblast generation, leading to multiorgan fibrosis and cardiac dysfunction in mice during aging. Mechanistically, SIRT6 bound to and repressed the expression of key TGF-ß signaling genes by deacetylating SMAD family member 3 (SMAD3) and Lys-9 and Lys-56 in histone 3. SIRT6 binding to the promoters of genes in the TGF-ß signaling pathway decreased significantly with age and was accompanied by increased binding of SMAD3 to these promoters. Our findings reveal that SIRT6 may be a potential candidate for modulating TGF-ß signaling to reduce multiorgan fibrosis during aging and fibrosis-associated diseases.

A nanopillar array on black titanium prepared by reactive ion etching augments cardiomyogenic commitment of stem cells.

A major impediment in the clinical translation of stem cell therapy has been the inability to efficiently and reproducibly direct differentiation of a large population of stem cells. Thus, we aimed to engineer a substrate for culturing stem cells to efficiently induce cardiomyogenic lineage commitment. In this work, we present a nanopillar array on the surface of titanium that was prepared by mask-less reactive ion etching. Scanning electron and atomic force microscopy revealed that the surface was covered by vertically aligned nanopillars each of ≈1 µm with a diameter of ≈80 nm. The nanopillars supported the attachment and proliferation of human mesenchymal stem cells (hMSCs). Cardiomyogenic lineage commitment of the stem cells was more enhanced on the nanopillars than on the smooth surface. When co-cultured with neonatal rat cardiomyocytes, the cyclic pattern of calcium transport observed distinctly in cells differentiated on the arrays compared to the cells cultured on the smooth surface was the functional validation of differentiation. The use of small molecule inhibitors revealed that integrins namely, α2ß1 and αvß3, are essential for cardiomyogenesis on the nanostructured surface, which is further mediated by FAK, Erk and Akt cell signaling pathways. This study demonstrates that the nanopillar array efficiently promotes the cardiomyogenic lineage commitment of stem cells via integrin-mediated signaling and can potentially serve as a platform for the ex vivo differentiation of stem cells toward cell therapy in cardiac tissue repair and regeneration.

SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity.

Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.

Sirtuin 6 mediated stem cell cardiomyogenesis on protein coated nanofibrous scaffolds.

The cellular niche provides combination of biomolecular and biophysical cues to control stem cell fate. Three-dimensional (3D) aligned nanofibrous scaffolds can effectively augment stem cell cardiomyogenesis. This work aims to understand the role of biomolecular signals from extracellular matrix (ECM) proteins and leverage them to further promote cardiomyogenesis on nanofibrous scaffolds. Human mesenchymal stem cells (hMSCs) were cultured on 3D aligned polycaprolactone scaffolds coated with different ECM proteins. Among multiple coatings tested, collagen coated fibers were most effective in promoting cardiomyogenesis as determined from increased expression of cardiac biomarkers and intracellular calcium flux. At molecular level, enhanced differentiation on collagen coated fibers was associated with an increased level of sirtuin 6 (SIRT6). Depletion of SIRT6 using siRNA attenuated the differentiation process through activation of Wnt signaling pathway. This study, thus, demonstrates that protein coated scaffolds can augment cardiomyogenic differentiation of stem cells through a combination of topographical and biomolecular signals.

Toll-like receptor 2 deficiency hyperactivates the FoxO1 transcription factor and induces aging-associated cardiac dysfunction in mice.

Toll-like receptors (TLRs) are a family of pattern-recognition receptors involved in innate immunity. Previous studies have shown that TLR2 inhibition protects the heart from acute stress, including myocardial infarction and doxorubicin-induced cardiotoxicity in animal models. However, the role of TLR2 in the development of aging-associated heart failure is not known. In this work, we studied aging-associated changes in structure and function of TLR2-deficient mice hearts. Whereas young TLR2-KO mice did not develop marked cardiac dysfunction, 8- and 12-month-old TLR2-KO mice exhibited spontaneous adverse cardiac remodeling and cardiac dysfunction in an age-dependent manner. The hearts of the 8-month-old TLR2-KO mice had increased fibrosis, cell death, and reactivation of fetal genes. Moreover, TLR2-KO hearts displayed reduced infiltration by macrophages, increased numbers of myofibroblasts and atrophic cardiomyocytes, and higher levels of the atrophy-related ubiquitin ligases MuRF-1 and atrogin-1. Mechanistically, TLR2 deficiency impaired the PI3K/Akt signaling pathway, leading to hyperactivation of the transcription factor Forkhead box protein O1 (FoxO1) and, in turn, to elevated expression of FoxO target genes involved in the regulation of muscle wasting and cell death. AS1842856-mediated chemical inhibition of FoxO1 reduced the expression of the atrophy-related ubiquitin ligases and significantly reversed the adverse cardiac remodeling while improving the contractile functions in the TLR2-KO mice. Interestingly, TLR2 levels decreased in hearts of older mice, and the activation of TLR1/2 signaling improved cardiac functions in these mice. These findings suggest that TLR2 signaling is essential for protecting the heart against aging-associated adverse remodeling and contractile dysfunction in mice.

Subcutaneous Ehrlich Ascites Carcinoma mice model for studying cancer-induced cardiomyopathy.

Cardiomyopathy is one of the characteristic features of cancer. In this study, we establish a suitable model to study breast cancer-induced cardiomyopathy in mice. We used Ehrlich Ascites Carcinoma cells to induce subcutaneous tumor in 129/SvJ mice and studied its effect on heart function. In Ehrlich Ascites Carcinoma bearing mice, we found significant reduction in left ventricle wall thickness, ejection fraction, and fractional shortening increase in left ventricle internal diameter. We found higher muscle atrophy, degeneration, fibrosis, expression of cell-adhesion molecules and cell death in tumor-bearing mice hearts. As observed in cancer patients, we found that mTOR, a key signalling molecule responsible for maintaining cell growth and autophagy was suppressed in this model. Tumor bearing mice hearts show increased expression and nuclear localization of TFEB and FoxO3a transcription factors, which are involved in the upregulation of muscle atrophy genes, lysosomal biogenesis genes and autophagy genes. We propose that Ehrlich Ascites Carcinoma induced tumor can be used as a model to identify potential therapeutic targets for the treatment of heart failure in patients suffering from cancer-induced cardiomyopathy. This model can also be used to test the adverse consequences of cancer chemotherapy in heart.

Systematic evaluation of the adaptability of the non-radioactive SUnSET assay to measure cardiac protein synthesis.

Heart is a dynamic organ that undergoes remodeling in response to both physiological and pathological stimuli. One of the fundamental cellular processes that facilitates changes in the size and shape of this muscular organ is the protein synthesis. Traditionally changes in cardiac protein synthesis levels were measured by radiolabeled tracers. However, these methods are often cumbersome and suffer from radioactive risk. Recently a nonradioactive method for detecting protein synthesis under in vitro conditions called the Surface Sensing of Translation (SUnSET) was described in cell lines of mouse dendrites and T cells. In this work, we provide multiple lines of evidence that the SUnSET assay can be applied to reliably detect changes in protein synthesis both in isolated neonatal primary cardiomyocytes and heart. We successfully tracked the changes in protein synthesis by western blotting as well as immunohistochemical variants of the SUnSET assay. Applying the SUnSET assay, we measured the cardiac protein synthesis during the different ages of mice. Further, we successfully tracked the increase in cardiac protein synthesis during different stages of a well-established model for pathological hypertrophy. Overall, we propose SUnSET assay as a simple, reliable and robust method to measure protein synthesis in the cardiac milieu.